Atrial natriuretic factor receptor on cultured Leydig tumor cells: ligand binding and photoaffinity labeling

Atrial natriuretic factor (ANF) is a peptide hormone discovered recently from the heart atrium that possesses potent natriuretic and vasorelaxant activities. Recently we found that ANF markedly stimulates intracellular cGMP and almost completely inhibits cAMP accumulation in testicular interstitial tumor cells [Pandey, K. N., Kovacs, W. J., & Inagami, T. (1985) Biochem. Biophys. Res. Commun. 133, 800-806]. These actions of ANF suggest the presence of ANF receptors in testicular interstitial cells. In this study, cultured murine Leydig tumor cells have been shown to contain specific binding sites for ANF. Saturation binding studies indicated a single class of binding sites with a Kd of 5 X 10(-9) M at a density of 2 X 10(6) sites/cell. The binding of mono[125I]iodo-ANF (125I-ANF) was competed by unlabeled ANF in a dose-dependent manner. Hormones unrelated to ANF such as angiotensin I, bovine luteinizing hormone, and human chorionic gonadotropin were not able to compete against 125I-ANF. The binding of 125I-ANF was rapid, reaching maximum levels in 15 min at 4 degrees C. At 37 degrees C, the cell-bound 125I label was quickly decreased. Pretreatment of cells with NH4Cl, chloroquine, or NaN3 resulted in significant increases in maximum levels of the cell-bound 125I radioactivity. A photoaffinity reagent for ANF receptor was prepared by reacting ANF with succinimido 4-azidobenzoate, and resultant 4-azidobenzoyl- (AZB-) ANF was purified by high-performance liquid chromatography (HPLC). AZB-ANF was radioiodinated by use of chloramine T and purified again by HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase.

We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37 degrees C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 +/- 0.077 nM and the Bmax. was 216 +/- 33 fmol bound/10(6) cells, which corresponds to 130,000 +/- 20,000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 microM-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increased the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demonstrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Comparison of binding and cyclic GMP accumulation by atrial natriuretic peptides in endothelial cells

Rat 125I-labeled atrial natriuretic factor (ANF (8-33)) was used to identify ANF receptors on cultured bovine aortic endothelial cells. Specific binding of 125I-ANF at 37 degrees C to confluent endothelial cells was saturable and of high affinity. Scatchard analysis of the equilibrium binding data indicated that endothelial cells contain a single class of binding sites with a Kd of 0.1 +/- 0.01 nM. This particular clone of endothelial cells had 16000 +/- 1300 receptors per cell. The order of potency for competing with 125I-ANF binding was human atrial natriuretic peptide (hANP) = atrial natriuretic factor (ANF (8-33)) greater than atriopeptin II greater than atriopeptin III greater than atriopeptin. The weakest competitor, atriopeptin I, had a K1 of 0.45 nM, which was only 6-fold higher than the K1 for hANP and ANF (8-33). ANF (8-33) and hANP in the presence of 0.5 mM isobutylmethyl-xanthine produced a 15-20-fold increase in cyclic GMP content at 10 pM and a maximal 500-fold elevation of cyclic GMP at 10 nM. The concentrations required to elicit a half-maximal increase in cyclic GMP for hANP, ANF (8-33), atriopeptin I, atriopeptin II and atriopeptin III were 0.30, 0.35, greater than 500, 4.0 and 5.0 nM, respectively. Although atriopeptin I acted as a partial agonist, it was unable to antagonize the effect of ANF (8-33) on cyclic GMP formation. These findings suggest that endothelial cells have multiple and functionally distinct ANF-binding sites.     

Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta

Specific binding site for atrial natriuretic factor (ANF), a potent natriuretic and vasorelaxant polypeptide recently isolated from mammalian atria, was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. Binding studies of 125I-labeled-synthetic alpha-human natriuretic peptide (alpha-hANP) revealed the presence of a non-interacting, single class of high affinity binding sites for alpha-hANP on VSMC in culture: the apparent dissociation constant (Kd) was approximately 1-2 X 10(-9)M and the number of maximal binding sites was approximately 200,000-300,000 sites/cell. A variety of vasoactive substances and other polypeptide hormones did not affect the binding of 125I-labeled-alpha-hANP to its binding sites. alpha-hANP significantly increased the concentrations of intracellular cyclic GMP in VSMC in a dose-dependent manner (3.2 X 10(-9)-1.6 X 10(-7)M). These data indicate that the specific receptor for ANF is present in VSMC and suggest that intracellular cyclic GMP may be involved in its vasorelaxant effect.     

Binding and internalization of atrial natriuretic factor by high-affinity receptors in A10 smooth muscle cells

A10 smooth muscle cells, derived from embryonic rat thoracic aorta, responded to the atrial natriuretic factor (ANF) with increased levels of cyclic GMP. These cells possess high-affinity (apparent Kd = 50 pM) plasma membrane receptors for ANF. Internalization of ANF at 37 degrees C was indicated by the following: approximately 25% of the 125I-ANF associated with the cells at elevated temperatures could not be dissociated from the surface of the cells, but could be released by permeabilization with saponin, and the amount of nondissociable ANF increased in the presence of chloroquine. In whole cells and in membranes, a single polypeptide of 60,000 Da was specifically labeled by a photoaffinity analog of 125I-ANF, as well as by crosslinking, and an IC50 of 80 pM for inhibition of the labeling by ANF was observed. The ANF receptor in A10 cells was distinguished from that in rabbit aorta by its high affinity for shorter and linear analogs of ANF, as well as by a different photolabeling pattern.     

Regulation of ANF receptor internalization: involvement of extracellular calcium.

Receptor mediated internalization of 125I-ANF (99-126) and the underlying mechanism was studied in PC12 cells. Phosphorylation of PC12 cell plasma membrane proteins at 0 degrees C or 37 degrees C was not altered in presence of ANF (99-126) or c-ANF (4-23). Exposure of cells to phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) did not alter the endocytic rate or extent of 125I-ANF (99-126) internalization. When cells were treated with a combination of PMA and the calcium ionophore A23187, internalization was not stimulated. Incubation with A23187 (10 microM) alone decreased 125I-ANF (99-126) internalization by 22% in Ca2+ containing medium. Cell surface binding increased 10% in the presence of Ca2+ compared to Ca2+ free medium, irrespective of the presence of A23187. Ca2+ appears to play an important role in the binding of ANF to the receptor and initiation of ligand-receptor complex internalization. Activation of protein kinase C or receptor phosphorylation is not an essential step in initiating ANF receptor internalization.     

Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells

In a previous study, we found that atriopeptin I was much weaker (EC50 greater than 500 nM) than atrial natriuretic factor (ANF-(8-33)) (EC50 = 0.3 nM) at increasing cyclic GMP in cultured endothelial cells. In this study, we used the cross-linking reagent disuccinimidyl suberate to investigate whether the differences in activity were due to the presence of multiple ANF receptors. When 98% of the ANF-binding sites on endothelial cells were occupied by tyrosine-atriopeptin I after cross-linking, there was no difference in the concentration-response curve to ANF-(8-33) with regard to cyclic GMP accumulation. In contrast, when 96% of the binding sites were occupied by cross-linked ANF-(8-33), a 60% decrease in the maximal cyclic GMP response was observed after the readdition of ANF-(8-33). These results suggest that ANF-(8-33) is binding to an additional site that atriopeptin I does not effectively bind. Affinity cross-linking of 125I-ANF to intact endothelial cells resulted in the labeling of two sites of Mr approximately 66,000 and approximately 130,000. Approximately 94% of the 125I-ANF binding sites had an Mr approximately 66,000. Labeling of this site was inhibited by both tyrosine-atriopeptin I (KI = 0.9 nM) and ANF-(8-33) (KI = 0.09 nM). Although 0.1 microM tyrosine-atriopeptin (AP I) inhibited labeling of the 66,000-dalton site to nearly the same degree as ANF-(8-33), it produced only a 4-fold increase in cyclic GMP compared to a 400-fold increase with ANF-(8-33). These results suggest that the 66,000-dalton site is not coupled to guanylate cyclase and cyclic GMP formation. Tyrosine-AP I (KI greater than 10 nM) was much weaker at competing for the 130,000-dalton site than ANF-(8-33) (KI = 0.075 nM). Because the EC50 for cyclic GMP stimulation for tyrosine-AP I (greater than 100 nM) and ANF-(8-33) (0.4 nM) is closer to the KI values for the 130,000-dalton protein, this site probably mediates the marked stimulation of cyclic GMP. Our results demonstrate that endothelial cells contain two binding sites for ANF-(8-33) and suggest that only the less abundant site (Mr approximately 130,000) is the receptor coupled to the activation of guanylate cyclase.     

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride

The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.     

Solubilization and molecular weight estimation of atrial natriuretic factor receptor from bovine adrenal cortex

Receptors for atrial natriuretic factor (ANF) have been solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate from bovine adrenal cortex and characterized. The detergent extract retained specific high-affinity binding sites for 125I-ANF. Scatchard analysis of the equilibrium binding data revealed a single class of binding site with a K-d of 1.8 nM and a maximum binding capacity of 2.5 pmol/mg of protein. The size of the 125I-ANF X receptor complexes was estimated to be 140,000 daltons by gel filtration on TSK gel G3000SW. Affinity labeling followed by electrophoresis under nonreducing conditions and autoradiography also revealed a single band of a similar size (Mr = 130,000); this band, however, migrated as a Mr = 70,000 species under reducing electrophoretic conditions. These results indicate that the ANF receptor, having a Mr of 130,000 - 140,000, is composed of disulfide-linked subunits and the ANF-binding site is located on the 70-kDa component.     

Identification of a receptor for atrial natriuretic factor in rabbit aorta membranes by affinity cross-linking

Binding sites in rabbit aorta membranes for atrial natriuretic factor (ANF) have been specifically and covalently labeled by two methods. In the first, the photoreactive analog of ANF, 125I-azidobenzoyl-ANF, was synthesized and used to photoaffinity label ANF receptors. In the second, 125I-ANF was covalently attached to its binding site by treatment of the 125I-ANF-receptor complex with bifunctional cross-linking agents. Analysis of the labeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that by both methods the same three protein bands were labeled. These bands had apparent molecular masses of 60,000, 70,000, and 120,000 daltons. With the photoaffinity label, half-maximal inhibition of labeling of each of these bands was achieved when approximately 200 pM of unlabeled ANF was included in the binding assay. These results suggest that these three different polypeptides are specific components of ANF receptors in rabbit aorta membranes.     

Identification of the receptor for atrial natriuretic factor on cultured vascular cells

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.     

Functional heterogeneity of atrial natriuretic factor receptor in bovine adrenal zona glomerulosa is explained by an amiloride-sensitive high affinity molecular complex

The effects of amiloride on the molecular characteristics of the atrial natriuretic factor (ANF) receptor from bovine adrenal zona glomerulosa were studied by computer modeling of competitive binding data, by affinity labeling experiments, and by steric exclusion high performance liquid chromatography of solubilized receptor. The order of potency of a series of truncated ANF analogs in competing for 125I-ANF binding to bovine adrenal zona glomerulosa membranes was the same as that obtained for inhibition of aldosterone secretion. Deletion of amino acids at the COOH-terminal end drastically reduced the affinities of the peptides. Computer analysis of competition curves revealed that all ANF analogs tested show similar binding characteristics: shallow competition curves, discrimination of varying proportions of high and low affinity binding states, and sensitivity to amiloride which increases the proportion of the high affinity binding component. These results from binding studies are suggestive of potential heterogeneity of ANF binding sites. In contrast, results from affinity cross-linking experiments are consistent with the notion of a single receptor protein. Incubation of membranes with increasing concentrations of 125I-ANF-(99-126) up to 3 nM resulted in the labeling of a single band of Mr 130,000. The ability of ANF analogs to compete for the labeling of the Mr 130,000 band by 125I-ANF-(99-126) agreed well with their potency as inhibitors of 125I-ANF binding to intact membranes. Addition of amiloride caused a dose-dependent increase in the labeling of the Mr 130,000 band. A single Mr 130,000 band was also labeled in bovine aorta and LLC-PK1 cell membranes. In order to further investigate the molecular basis for the apparent heterogeneity of ANF binding we have prelabeled the membrane receptor with 125I-ANF-(99-126) prior to solubilization with octyl-beta-D-glucoside and chromatography on a Superose 6 steric exclusion column. The elution profile of the prelabeled receptor consistently showed two peaks of radioactivity with mean Stokes radii of 70 and 50 A. When amiloride was added to the incubation medium, the elution profile consisted almost exclusively of the 70-A peak. Quantitative analysis of the chromatographic profiles revealed that amiloride increases by 2-3 times the area of the 70-A peak. We conclude that the 70-A form represents a ternary complex of the receptor with an amiloride-sensitive effector protein.     

Identification and characterization of atrial natriuretic factor receptors in the rat retina

The characteristics of atrial natriuretic factor (ANF) receptors where studied in rat retinal particulate preparations. Specific 125I-ANF binding to retinal particulate preparations was greater than 90% of total binding and saturable at a density (Bmax) of 40 +/- 8 fmol/mg protein with an apparent dissociation constant (Kd) of 6.0 +/- 2.0 pM (n = 3). Apparent equilibrium conditions were established within 30 min. The Kd value of 125I-ANF binding calculated by kinetic analysis was 4.0 pM. The Bmax of 60 +/- 10 fmol/mg protein and the Kd of 5 +/- 2 pM, calculated by competition analysis, were in close agreement with the values obtained from Scatchard plots or kinetic analysis. The 125I-ANF binding to retinal particulate preparations was not inhibited by 1 microM concentration of somatostatin, vasopressin, vasoactive intestinal peptide, adrenocorticotropin, thyrotropin releasing hormone, or leu-enkephalin. The rank order of potency of the unlabelled atrial natriuretic peptides for competing with specific 125I-ANF (101-126) binding sites was rANF (92-126) greater than rANF (101-126) greater than rANF (99-126) greater than rANF (103-126) greater than Tyro-Atriopeptin I greater than hANF (105-126) greater than rANF (1-126). Similar results have been obtained in peripheral tissues and mammalian brain, indicating that central and peripheral ANF-binding sites have somewhat similar structural requirements. Affinity cross-linking of 125I-ANF to retinal particulate preparations resulted in the labelling of two sites of molecular weight 140 and 66 kDa, respectively. This demonstration of specific high-affinity ANF receptors suggests that the peptide may act as a neurotransmitter or neuromodulator in the retina.     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.     

Atrial natriuretic factor regulation of cyclic GMP levels and steroidogenesis in isolated fasciculata cells of rat adrenal cortex

Isolated fasciculata cells of rat adrenal cortex, when incubated with atrial natriuretic factor (ANF), stimulated the levels of cyclic GMP and corticosterone production in a concentration-dependent manner without a rise in the levels of cyclic AMP. The ANF-dependent elevation of cyclic GMP was rapid, with a detectable increment in 30 s. ANF also stimulated the particulate guanylate cyclase. These results not only indicate the coupling of cyclic GMP and corticosterone production with ANF signal, but also demonstrate that, like the ACTH signal, cyclic AMP is not the mediator of ANF-induced adrenocortical steroidogenesis.     

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.